Abstract: Nearly half of the world's population lives in countries where malaria is endemic. Plasmodium falciparum, the causative agent of the most severe form of human malaria, is responsible for 95% of malaria deaths worldwide. This project's main goals are to characterize the molecular determinants that control chromatin organization and gene regulation in this pathogen; elucidate their importance in parasite development, virulence, and sexual differentiation; and identify novel pathways that can be targeted to kill the parasite. The proposed research builds upon a large body of molecular, cellular, and genome-wide data generated in the Pl's lab that discovered how close interconnections between 3D genome organization and transcription regulate sexual differentiation and parasite development. Despite significant progress in the past 10 years in elucidating the role of chromatin in transcriptional control, the mechanism underlying changes in chromatin structure and the factors controlling these changes remain to be elucidated. The studies proposed here will examine the proteins and long non-coding RNAs (lncRNAs) involved in the control of chromatin structure, parasite development, virulence, and sexual differentiation. The project is organized into three Specific Aims. Aim 1 will validate the role and essentiality of two proteins that we previously identified as potential master regulators of male and female sexual differentiation. Two genes encoding an AP2 transcription factor (PfApi2AP2-FG) and a serine/threonine protein phosphatase 2A activator (PfPTPA) are located at the boundary of two condensed chromatin super domains observed in both early and late gametocytes. We will fully investigate the function of these proteins during male and female gametocyte development using cellular, molecular, and genetic approaches. Aim 2 will validate the role of two lncRNAs (lncRNAG9 and lncRNAG14) that we previously identified as potential regulators of sexual differentiation and chromosome reorganization during parasite development. These factors may be involved in chromosome reorganization in gametocyte stages. We will therefore implement a set of cellular, molecular, and genetic approaches to characterize their role in the formation and maintenance of parasite sexual stages, as well as in the specific chromosomal reorganization we observed at these stages. Aim 3 will systematically isolate and identify the proteins and lncRNAs that control the structure and activity of transcriptionally silent heterochromatin clusters in P. falciparum genome, using a novel methodology called chromatin isolation by RNA purification (ChlRP). Once identified, factors will be validated at the functional level using cellular and molecular experimental approaches, including genome editing by CRlSPR-Cas9. lt is anticipated that the proposed research will offer groundbreaking new insights into parasite-specific protein complexes and lncRNAs and their role in chromatin structure and parasite biology, as well as many starting points for novel directions in malaria research and therapy.